Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells.

We have examined the interaction between muscarinic and alpha(2)-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca(2+) concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca(2+) and IP formation, activation of alpha(2)-receptors does not significantly increase either intracellular Ca(2+) or IPs over basal levels. However, simultaneous activation of muscarinic and alpha(2)-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca(2+) increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and alpha(2)-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca(2+), because it is decreased but not prevented by blocking the entry of Ca(2+) with LaCl(3) or chelating intracellular Ca(2+) with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca(2+) resulting from simultaneous activation of muscarinic and alpha(2)-adrenergic receptors.

Synergistic receptor-activated calcium increases in single nonpigmented epithelial cells.

PURPOSE: To determine whether single nonpigmented ciliary body cells contain the signaling mechanism to produce synergistic drug-activated increases in Ca2+, or whether these responses are produced cooperatively by interaction among groups of cells. METHODS: Suspensions of single nonpigmented cells were plated onto soft collagen gels. Fura-2 fluorescence ratio imaging was used to examine receptor-evoked changes in intracellular Ca2+ concentration. RESULTS: Nonpigmented cells plated on soft collagen gels retained a rounded shape with membrane evaginations visible on their surface. Application of acetylcholine (10 microM) or epinephrine (1 microM) each produced small increases in intracellular Ca2+, but in combination they produced a Ca2+ increase of more than 10-fold. This synergistic Ca2+increase was a result of activation of muscarinic and alpha2-adrenergic receptors because a specific alpha2-adrenergic agonist could substitute for epinephrine in producing the response. The response could be blocked by a specific alpha2-antagonist and a muscarinic antagonist. An alpha1-agonist could not substitute for epinephrine in producing a synergistic increase nor could the synergism be blocked by alpha1- or beta-antagonists. The Ca2+ increase was largely produced by release from internal stores, because the peak amplitude of the response was nearly the same in the external solution containing a low Ca2+ concentration; however, the influx of Ca2+ into the cell was responsible for maintenance of a steady component of the Ca2+ increase during maintained drug stimulation and for refilling the internal stores. CONCLUSIONS: Single nonpigmented cells can produce synergistic increases in Ca2+ on multiple receptor activation, indicating that the mechanism of synergism does not require the interaction of multiple cells. The Ca2+ increase is a result of release from internal stores and Ca2+ entry through an as yet undefined conductance or transport system in the plasma membrane.

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts.

We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca2+ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca2+ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca2+ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell's cytoarchitecture.

Synergistic rise in Ca2+ produced by somatostatin and acetylcholine in ciliary body epithelial cells.

The purpose of these experiments was to demonstrate the presence of somatostatin receptors on the nonpigmented epithelial cells of the rabbit ciliary body and their link with intracellular Ca2+ homeostasis. Freshly excised rabbit ciliary processes and nonpigmented cell layer, explants were loaded with the fluorescent dye fura-2, and free-Ca2+ concentration ([Ca2+]i) in the nonpigmented cells was measured with fluorescence ratio imaging. The cells were continuously perfused, and drugs were added to the perfusate. Somatostatin-14 (SS14, 0.1-1.0 microM) or acetylcholine (ACh, 10 microM) applied alone produced small increases in [Ca2+]i. However, SS14 (0.1 microM) in combination with ACh (10 microM) induced a massive increase in [Ca2+]i (25.7 +/- 3.3 times the baseline level, n = 28). The dose-response curve for SS14 (in the presence of 10 microM ACh) was sigmoidal with an EC50 of 3.9 nM and Hill coefficient of 2.5, indicating the requirement for multiple SS receptor activation. Somatostatin-28 could mimic the effect of SS14, although a much higher concentration was required. Shifting the SS14 dose-response curve to the right by about two-orders of magnitude resulted in a fit to the SS28 data. The response to ACh + SS14 could not be blocked by the alpha 2-adrenergic blocker yohimbine (Yoh, 10 microM) or the A1-specific adenosinergic antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 microM). Incubation of the tissue with pertussis toxin (PTx, 1 microgram ml-1) did not alter the response to ACh alone but eliminated the synergistic effect of somatostatin. We conclude that nonpigmented epithelial cells of the rabbit ciliary body possess a novel somatostatin receptor whose activation can synergistically potentiate the rise in [Ca2+]i produced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through Gi.

Synergistic increase in Ca2+ produced by A1 adenosine and muscarinic receptor activation via a pertussis-toxin-sensitive pathway in epithelial cells of the rabbit ciliary body.

The combined effects of adenosine and acetylcholine on the intracellular free-Ca2+ concentration in nonpigmented epithelial cells of the rabbit ciliary body were investigated using fura-2 fluorescence-ratio imaging. Acetylcholine (10 microM) by itself produced a modest increase in [Ca2+]i. Acetylcholine in combination with adenosine, or with the A1-specific agonists N6-cyclohexyl-adenosine, N6-cyclopentyl-adenosine and (R)-N6-(2-phenyl-1-methylethyl)-adenosine (0.1-1 microM), induced a massive increase in [Ca2+]i, which could be blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine. However, the A2-specific agonist 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamide-ade nos ine and the antagonist 3,7-dimethyl-1-(2-propynyl)xanthine were without effect. Incubation of the tissue with pertussis toxin did not alter the response to ACh alone but eliminated the synergistic effect of adenosine (or of epinephrine). It was concluded that in the epithelial cells of the rabbit ciliary body, adenosine and epinephrine synergistically potentiate the rise in [Ca2+]i produced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through G(i).

Synergistic effect of adrenergic and muscarinic receptor activation on [Ca2+]i in rabbit ciliary body epithelium.

1. Changes in cytosolic free calcium concentration ([Ca2+]i) in response to cholinergic and adrenergic agents alone and in combination were investigated using fura-2 fluorescence imaging in intact non-pigmented epithelial cells of rabbit ciliary body. 2. Resting ('baseline') [Ca2+]i was 147 +/- 6 nM (mean +/- S.E.M.). Acetylcholine (ACh, 10 microM) doubled [Ca2+]i, and adrenaline (1 microM) increased it by about 36%. When ACh (10 microM) and adrenaline (1 microM) were applied together [Ca2+]i was transiently increased to 1160 +/- 160 nM, about 7 times the response induced by ACh alone. 3. Noradrenaline and 5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline (UK 14304) had effects similar to adrenaline in enhancing the response to ACh. Phenylephrine (Phe) had a relatively smaller effect and none was observed for methoxamine and isoprenaline (Iso). 4. The response to ACh and adrenaline could be blocked by atropine (1 microM, 87 +/- 5%), yohimbine (1 microM, 73 +/- 8%), and to a lesser degree by prazosin (1 microM). Propranolol had no effect. 5. Lowering the extracellular calcium concentration to 3 nM dropped the baseline [Ca2+]i by half and reduced the response to ACh and adrenaline to a small and transient rise in [Ca2+]i. Addition of La3+ to Ca(2+)-containing solution also lowered [Ca2+]i and largely reduced the response. 6. We conclude that simultaneous activation of muscarinic and alpha 2-adrenergic receptors induces a large increase in [Ca2+]i, which is the result of both Ca2+ release and influx.

Dihydropyridine-sensitive Ca2+ spikes and Ca2+ currents in rabbit ciliary body epithelial cells.

Intracellular microelectrode and whole-cell patch-clamp recordings were used to investigate a Ba(2+)-induced regenerative depolarization and its underlying Ba2+ current in the ciliary body epithelial cells of the rabbit eye. Exposure of these epithelial cells to 4-10 mmol l-1 Ba2+ depolarized the membrane potential and caused the generation of one or more spikes, before the membrane potential reached a steady-state level. The spikes, but not the slow phase of depolarization, could be blocked with Co2+ (2 mmol l-1), Gd3+ (25 mumol l-1), La3+ (20 mumol l-1), Cd2+ (10 mumol l-1), verapamil (30 mumol l-1) and nifedipine (1 mumol l-1). Tetrodotoxin at 100 nmol l-1 had no effect. In the absence of Na+, but in the presence of external Ba2+, step depolarization of the membrane potential activated an inward current that could be blocked with Co2+ (2 mmol l-1), Cd2+ (10 mumol l-1) and nifedipine (1 mumol l-1), but not with Ni2+ (50 mumol l-1) or omega-conotoxin (1-10 mumol). This inward current could be enhanced with the dihydropyridine agonist (+/-)BAY K 8644 (1 mumol l-1). The inactivation characteristics of the inward current (v1/2 = -38.7 mV, k = 12.6 mV) is most like that seen in neurons. These findings indicate that the epithelial cells of the ciliary body possess dihydropyridine-sensitive, voltage-activated Ca2+ channels.

Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials.

Volume regulation of non-pigmented cells from ciliary epithelium.

We describe a new method for investigating ion and water transport in ciliary epithelium. A single ciliary process from the rabbit is isolated, placed in a chamber, and rapidly perfused with physiological Ringer. If this process is then viewed at its edge with a light microscope using Hoffman modulation contrast optics, it is possible to record the image of a single layer of non-pigmented epithelial cells. When these cells are exposed to hypotonic Ringer, they swell and then reduce their volume by extruding salt and water. We have used the rate of swelling to calculate the hydraulic conductivity of the non-pigmented cells. These measurements show that the aqueous humor could be secreted by the generation of a modest osmotic gradient across the non-pigmented cell basal membrane. We have also used this preparation to investigate the mechanism of the decrease in cell volume after swelling in hypotonic medium. This volume regulatory decrease can occur at a rate as large as the rate of normal fluid transport during the secretion of the aqueous. It appears to be caused in part by active Na extrusion, since it is partially inhibited by exposure to ouabain. In addition, there appears also to be a contribution to the regulatory response from a swelling-induced K efflux, since the rate of volume regulation can be altered by perfusion with Ba2+ and by changing the extracellular K concentration.